Mann M B, Rao R N, Smith H O
Gene. 1978 Apr;3(2):97-112. doi: 10.1016/0378-1119(78)90054-9.
The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R-PstI site, (formula: see text), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3'-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3'-OH termini, thus regenerating the PstI cleavage site sequences. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r-m-recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage lambdacI-0). A single phage-resistant clone was found. The recombinant plasmid, pD110, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.
溶血嗜血杆菌II类限制修饰系统（HhaII）的基因已在大肠杆菌中克隆。用于克隆的载体是质粒pBR322，它赋予对四环素和氨苄青霉素的抗性，并且在氨苄青霉素基因中含有单个内切酶R-PstI位点（分子式：见正文）。Bolivar等人（1977年）开发的方法用于形成DNA重组体。溶血嗜血杆菌DNA用PstI内切酶切割，使用末端脱氧核苷酸转移酶将聚（dC）延伸物添加到3'-OH末端。环状pBR322 DNA用PstI内切酶切割成线性分子，并将聚（dG）延伸物添加到3'-OH末端，从而再生PstI切割位点序列。通过使两种DNA退火形成的重组分子用于转染大肠杆菌的限制和修饰缺陷菌株（HB101 r-m-recA）。测试四环素抗性克隆是否获得限制表型（通过在接种有噬菌体λcI-0的平板上生长来测量）。发现了一个抗噬菌体克隆。从该克隆中分离出的重组质粒pD110获得了3千碱基的额外DNA，其可以用PstI内切酶切除。除了限制功能外，携带该质粒的细胞还表达HhaII修饰功能。两种活性都通过单链DNA-琼脂糖色谱法进行了部分纯化。克隆的HhaII限制活性产生与HinfI相同的切割模式。给出了克隆DNA片段的限制图谱。
